Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive. Both SecA and the ribosome need to interact with the translocon during membrane protein insertion. SecA competes with ribosomes and ribosome-nascent chain complexes for binding to the translocon. SecA and ribosome binding to the translocon is mutually exclusive, implying that during membrane protein insertion, both ligands bind the translocon in a sequential manner. Insight in the mechanism of membrane protein insertion.